Elucidating the molecular events that promote tumor cell invasion continues to be a challenge for the treatment and prevention of hepatocellular carcinoma (HCC). HCC is a highly aggressive carcinoma of the liver which has been reported to occur world-wide in increasing numbers. Intra-hepatic metastatic recurrences via the portal vein are the main cause of death in HCC patients who have undergone partial hepatectomy or liver transplantation (Korn, World J. Gastroenterol., 7:777-8, 2001). Identifying the main ‘players’ and how they contribute to the tumor cell metastatic cascade, for example at the early stages of cellular invasion, can present opportunities for lessening the severity of HCC through new therapeutic interventions.
Osteopontin (OPN, SPP1) is a secreted multi-functional glycoprotein expressed at high levels in tumors and the surrounding stroma of numerous cancers, including those of the liver, and is alternatively spliced into at least 3 isoforms (OPN-a, OPN-b, OPN-c) (Butler, Ann. N.Y. Acad. Sci., 760:6-11, 1995; Coppola et al., Clin. Cancer Res., 10:184-90, 2004). Increased serum and plasma OPN levels (˜4-10 fold) are associated with advanced stage lung, hepatic, breast, colon, and prostate carcinomas (Oates et al., Invasion Metastasis, 17:1-15, 1997; Fedarko et al., Clin. Cancer Res., 7:4060-6, 2001; Singhal et al., Clin. Cancer Res., 3:605-11, 1997.). OPN expression can predict high grade, late stage and early recurrence HCC (Pan et al., Cancer, 98:119-27, 2003) and is highly correlated with tumor recurrence and decreased patient survival following orthotopic liver transplantation (Wang, Hepatology, 42 (4), Suppl. 1:391A, 2005). Prominent OPN levels have been detected in metastatic HCC tumor cells at the leading edge of pseudopodia and filopodia (Suzuki et al., J. Bone Miner. Res., 17:1486-97, 2002) and in macrophages at the tumor-stroma interface (Senger et al., Ann. N.Y. Acad. Sci., 760: 83-100, 1995). A correlation between OPN mRNA expression and primary HCC tumor metastasis has been shown (Ye et al., Nat. Med., 9: 416-423, 2003). Cytoplasmic OPN was detected in vascularized regions of primary HCC tumors but not in normal liver. In addition, a neutralizing antibody to OPN decreased pulmonary secondary lesions in nude mice and inhibited tumor cell invasion.
Local and extra-hepatic HCC tumor cell invasion is associated with extensive matrix remodeling, angiogenesis, and hepatocyte injury (McKenna et al., Am. J. Surg., 183: 588-94, 2002). The members of the zinc-dependent endopeptidase family of matrix metalloproteinases (MMPs) catabolize extracellular matrix components (Theret et al., Hepatology, 34:82-88, 2001; Liaw and Crawford, Braz. J. Med. Biol. Res., 32:805-812, 1999). Each MMP contains a catalytic and pro-peptide regulatory domain and a variable number of carboxy-terminal hemopoexin-like structural domains and are broadly divided into subclasses based on substrate activity. The two gelatinases (MMP-2 and MMP-9) play roles in tumor invasion and angiogenesis and participate in cancer progression in several neoplasias (Turpeenniemi-Hujanen, Biochimie, 87:287-97, 2005; Hanemaaijer et al., Int. J. Cancer, 86:204-7, 2000; Scorilas et al., Br. J. Cancer, 84:1488-96, 2001). Active MMP-9 enzymatically cleaves proteins of the basement membrane (such as collagens type IV, V, VII, X, and XIV) and can be detected at the invasive front of HCC (Kaneyoshi et al., Clin. Cancer Res., 7:4027-32, 2001). A substantial increase in MMP-9 mRNA levels in HCC primary metastatic tumors has been observed (Ye et al., Nat. Med. 9:416-23, 2003), which is consistent with indications of HCC tumor malignancy and MMP-9 abundance (Ashida et al., Am. J. Pathol., 149:1803-11, 1996; Wei et al., Hunan. Yi. Ke. Da. Xue. Xue. Bao., 28:212-6, 2003).
Stromelysin-1 (MMP-3) and matrilysin (MMP-7) are reported to cleave OPN at residues 166 and 210 (Agnihotri et al., J. Biol. Chem., 276:28261-7, 2001). MMP-3/-7 digested OPN fragments increase AsPC-1 and HeLa tumor cell adhesion via cell surface integrin receptors and MMP-3 cleaved OPN can increase mouse peritoneal macrophage cell migration. The thrombin coagulation factor also cleaves OPN at residue 168, resulting in two fragments of similar molecular weight (˜28-30kD) that can be detected in the serum and plasma of patients with cancer (Senger et al., Cancer Res., 48:5770-4, 1988). Thrombin-cleaved OPN can mediate increased tumor and macrophage cell adhesion and migration via exposure of the amino-terminal reactive RGD sequence and binding to cell surface integrins, namely the vitronectin receptor αVβ3, although interactions with αVβ1, αVβ5, α4β1, and α9β1 have been described (Sodek et al., Crit. Rev. Oral Biol. Med., 11:279-303, 2000; Wai and Kuo, J. Surg. Res., 121:228-41, 2004; Weber, Biochim. Biophys. Acta, 1552:61-85, 2001). Conversely, the COOH-terminal thrombin-cleaved fragment has been proposed to induce macrophage migration primarily through CD44 receptors (Weber et al., J. Leukoc. Biol., 72:752-61, 2002).